Methods of determination of activation or inactivation of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) hormonal systems

ABSTRACT

An in vitro method of determining activation or inactivation of the atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) hormonal systems, the method comprising simultaneously detecting the presence or amount of atrial and brain natriuretic peptide prohormones (proANP and proBNP) or fragments thereof in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §371 from internationalapplication PCT/EP2004/006971, filed Jun. 28, 2004, which claimspriority from United Kingdom Application Serial No. 0315291.5, filedJun. 30, 2003.

TECHNICAL FILED OF THE INVENTION

The invention relates to test methods useful for the diagnosis and/ormonitoring treatment of cardiac conditions such as heart failure and tosubstances for use in the methods.

BACKGROUND OF THE INVENTION

Congestive heart failure (CHF) is a clinical syndrome caused by heartdisease, characterised by breathlessness and abnormal sodium and waterretention, and resulting in oedema. This occurs when the heart is unableto generate a cardiac output sufficient to meet the demands of the bodywithout marked increase of diastolic pressure. It is a consequence of acardiac disease which impairs ventricular systolic or diastolicfunction, or both. It is not a single disease but the end stage of manydifferent forms of heart diseases, the most common of which are thecoronary artery diseases, hypertension and diabetes (Kannel et al.1994). Heart failure is manifested by symptoms of poor tissue perfusion(e.g., fatigue, poor exercise tolerance) or congestion of vascular beds(e.g., dyspnoea, pulmonary oedema, peripheral oedema) or both. Treatmentof heart failure is generally directed towards its underlying causes.

The prevalence of symptomatic heart failure in the general population inEurope is estimated to be about 0.4-2%. As the prevalence rises rapidlywith age, the increasing life expectancy is expected to have a majorimpact on the incidence of heart failure in the near future. Theasymptomatic form of left-ventricular systolic dysfunction is estimatedto be as common as symptomatic congestive heart failure (McDonagh et al.1997).

The current routine clinical and investigative parameters used for thediagnosis of heart failure (clinical examination, electrocardiography,chest X-ray) have been found to be inadequate because the diagnosiscauses false-positive results (Remes et al. 1991). Echocardiographyprovides specific diagnostic and prognostic information, but it is notparticularly suitable for screening or for rapid point-of-carediagnostics. Thus, there is a need for new diagnostic tests for cardiacimpairment.

A number of studies have demonstrated the usefulness of measurement ofsingle peptides derived from atrial natriuretic peptide prohormone(proANP) and brain natriuretic peptide prohormone (proBNP) in thediagnosis of heart failure (Talwar et al. 2000; De Lemos et al. 2001;Daly et al. 2002). Cardiac impairment is associated with elevatedcirculating levels of atrial natriuretic peptide (ANP), brainnatriuretic peptide (BNP), N-terminal fragment of proANP (NT-proANP) andN-terminal fragment of proBNP (NT-proBNP) (Sagnella 1998). High plasmaconcentrations correlate with poor prognosis after myocardial infarctionand heart failure (Omland et al. 2002). Moreover, monitoring plasmalevels of NT-proBNP appears to offer more powerful guidance in therapyof heart failure than follow-up by conventional clinical parameters(Troughton et al. 2000).

However prior art diagnostic methods, such as those disclosed in WO87/06938, WO 00/35951, WO 91/00292, U.S. Pat. No. 5,786,163, EP 648 228B1, WO 00/45176, WO 00/19207, U.S. Pat. No. 6,124,430, EP 542 255 B1) orthose commercially available, are only intended to measure, and are onlycapable of measuring, a single peptide (ANP, BNP, NT-proANP orNT-proBNP) at a time. For example, the prior art discloses the use ofANP receptor or NPRA (GC-A) receptor in assays to determine natriureticpeptides, but does not disclose any simultaneous determination ofnatriuretic peptides. U.S. Pat. No. 5,747,274 discloses simultaneousdetection of at least three cardiac markers using at least threedifferent monoclonal or polyclonal antibody pairs, each specific for adifferent marker. Consequently, these assays produce multiple results.Thus there remains in the art a need for a reliable and sensitive butrelatively cheap and simple means for detecting or diagnosing cardiacimpairment such as heart failure.

Accordingly the present invention provides a test method which detectsactivation or inactivation of the ANP and BNP hormonal systems byassaying for both proANP- and proBNP-derived peptides simultaneously.Both proANP and proBNP derived peptides may be assayed in the samesample, at the same time. The method produces a single result and issimpler to perform than prior art methods. Moreover the present assaymethods show greater sensitivity than prior art methods. Further still,the present test has a profound capability to give a reliable testresult whether the patient is in an early phase or late phase of heartfailure. The single assay format of the present invention, performedsimultaneously per se, offers a cheaper and more cost effectivealternative to the available tests thus allowing reliable measurement ofactivation or inactivation of both the ANP and the BNP hormonal systems

SUMMARY OF THE INVENTION

Accordingly the present invention provides an in vitro method ofdetermining activation or inactivation of the atrial natriuretic peptide(ANP) and brain natriuretic peptide (BNP) hormonal systems, the methodcomprising simultaneously detecting the presence or amount of atrial andbrain natriuretic peptide prohormones (proANP and proBNP) or fragmentsthereof in a sample.

The invention also provides:

an agent which comprises:

-   -   (a) (i) proANP (SEQ ID NO. 1), ANP (SEQ ID NO. 2) or NT-proANP        (SEQ ID NO. 3);        -   (ii) a homologous sequence having at least 70% identity to            (i); or        -   (iii) a fragment of (i) or (ii) which is at least 6 amino            acids in length; and    -   (b) (i) pro-BNP (SEQ ID NO. 4), BNP (SEQ ID NO. 5), NT-proBNP        (SEQ ID NO. 6);        -   (ii) a homologous sequence having at least 70% identity to            (i); or        -   (iii) a fragment of (i) or (ii) which is at least 6 amino            acids in length;

a polynucleotide comprising sequence which encodes the agent, orcomplementary sequence:

an expression vector and host cell comprising the polynucleotide;

a process for producing the polypeptide agent which comprises:

(a) cultivating the host cell under conditions to provide for expressionof the polypeptide; and optionally

(b) recovering the expressed polypeptide;

a method of identifying a substance that binds specifically to

(a) (i) proANP (SEQ ID NO. 1), ANP (SEQ ID NO. 2) or NT-proANP (SEQ IDNO. 3);

-   -   (ii) a homologous sequence having at least 70% identity to (i);        or    -   (iii) a fragment of (i) or (ii) which is at least 6 amino acids        in length and

(b) (i) pro-BNP (SEQ ID NO. 4), BNP (SEQ ID NO. 5), NT-proBNP (SEQ IDNO. 6);

-   -   (ii) a homologous sequence having at least 70% identity to (i);        or    -   (iii) a fragment of (i) or (ii) which is at least 6 amino acids        in length which method comprises:

(A) contacting a candidate substance with (a) and (b) under conditionswhich allow specific binding; and

(B) determining whether the candidate substance binds to (a) and (b);

an antibody, fragment or derivative thereof which is able to bind toboth:

(a) (i) proANP (SEQ ID NO. 1), ANP (SEQ ID NO. 2) or NT-proANP (SEQ IDNO. 3);

-   -   (ii) a homologous sequence having at least 70% identity to (i);        or    -   (iii) a fragment of (i) or (ii) which is at least 6 amino acids        in length; and

(b) (i) pro-BNP (SEQ ID NO. 4), BNP (SEQ ID NO. 5) or NT-proBNP (SEQ IDNO. 6);

-   -   (ii) a homologous sequence having at least 70% identity to (i);        or    -   (iii) a fragment of (i) or (ii) which is at least 6 amino acids        in length

a process for making the antibody;

a solid support comprising the antibody.

The invention further provides methods of diagnosing and/or monitoringtreatment of heart failure and a diagnostic kit for use in such methods.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Purification by reverse phase HPLC of a novel protein or peptideagent of the invention.

FIG. 2 a: A competitive binding curve for immunoassay of NT-proXNP.

FIG. 2 b: Development of antibody titres in immunisation of a goat usinga GST-fusion protein of NT-proXNP2 as immunogen.

FIG. 3: Serum levels of NT-proANP, NT-proBNP and NT-proXNP in patientswith cardiac disorders.

FIG. 4: Serum levels of NT-proANP, NT-proBNP and NT-proXNP in cardiacpatients.

FIG. 5: Response of plasma NT-proANP, NT-proBNP and NT-proXNP andcardiac output (CO) in patients of heart failure to therapy.

BRIEF DESCRIPTION OF THE SEQUENCES

-   -   SEQ ID NO: 1 amino acid sequence of human proANP    -   SEQ ID NO: 2 amino acid sequence of human ANP    -   SEQ ID NO: 3 amino acid sequence of human NT-proANP    -   SEQ ID NO: 4 amino acid sequence of human proBNP    -   SEQ ID NO: 5 amino acid sequence of human BNP    -   SEQ ID NO: 6 amino acid sequence of human NT-proBNP    -   SEQ ID NO: 7 nucleotide sequence encoding human proANP    -   SEQ ID NO: 8 nucleotide sequence encoding human ANP    -   SEQ ID NO: 9 nucleotide sequence encoding human NT-proANP    -   SEQ ID NO: 10 nucleotide sequence encoding human proBNP    -   SEQ ID NO: 11 nucleotide sequence encoding human BNP    -   SEQ ID NO: 12 nucleotide sequence encoding human NT-proBNP    -   SEQ ID NO: 13 amino acid sequence of an agent according to the        invention NT-proXNP1    -   SEQ ID NO: 14 amino acid sequence of an agent according to the        invention NT-proXNP2    -   SEQ ID NO: 15 amino acid sequence of an agent according to the        invention NT-proXNP3    -   SEQ ID NO: 16 amino acid spacer sequence    -   SEQ ID NO: 17 amino acid sequence of an agent according to the        invention NT-proXNP4    -   SEQ ID NO: 18 amino acid sequence of an agent according to the        invention NT-proXNP5    -   SEQ ID NO: 19 amino acid sequence of an agent according to the        invention proXNP6    -   SEQ ID NO: 20: amino acid sequence of an agent according to the        invention XNP7    -   SEQ ID NO: 21 nucleotide sequence encoding NT-proXNP1    -   SEQ ID NO: 22 nucleotide sequence encoding NT-proXNP2    -   SEQ ID NO: 23 nucleotide sequence encoding NT-proXNP3    -   SEQ ID NO: 24 nucleotide sequence encoding NT-proXNP4    -   SEQ ID NO: 25 nucleotide sequence encoding NT-proXNP5    -   SEQ ID NO: 26 nucleotide sequence encoding proXNP6    -   SEQ ID NO: 27 nucleotide sequence encoding XNP7    -   SEQ ID NO: 28 primer sequence    -   SEQ ID NO: 29 primer sequence    -   SEQ ID NO: 30 primer sequence    -   SEQ ID NO: 31 primer sequence    -   SEQ ID NO: 32 primer sequence    -   SEQ ID NO: 33 amino acid sequence of human GC-A receptor    -   SEQ ID NO: 34 amino acid sequence of extracellular domain of        human GC-A receptor    -   SEQ ID NO: 35 amino acid sequence of human GC-B receptor    -   SEQ ID NO: 36 amino acid sequence of human GC-C receptor

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a new test method which is useful fordiagnosing and/or monitoring treatment of cardiac disease, in particularheart failure and to components and kits for use in the method. Themethod allows the detection of activation or inactivation of the atrialnatriuretic peptide (ANP) hormonal system and the brain natriureticpeptide (BNP) hormonal system in an individual simultaneously. Asingle-test method may be used. In general the method assayssimultaneously for the presence and/or amount of peptides derived fromA- and B-type natriuretic peptide prohormones in a suitable biologicalsample obtained from the individual.

TERMS AND ABBREVIATIONS

-   proANP is atrial natriuretic peptide prohormone;-   proANP is processed by cleavage of the N-terminal fragment into the    mature atrial natriuretic peptide (ANP). Human proANP has 126 amino    acids (proANP₁₋₁₂₆)

SEQ ID NO: 1 NPMYNAVSNA DLMDFKNLLD HLEEKMPLED EVVPPQVLSE PNEEAGAALSPLPEVPPWTG EVSPAQRDGG ALGRGPWDSS DRSALLKSKL RALLTAPRSL RRSSCFGGRMDRIGAQSGLG CNSFRY

-   ANP is atrial natriuretic peptide-   Human ANP is formed by amino acids 99 to 126 of the prohormone    (proANP₉₉₋₁₂₆)

SEQ ID NO: 2 SLRRSSCFGG RLMDRIGAQSG LGCNSFRY

-   NT-proANP is the N-terminal fragment of proANP-   The N-terminal fragment of human proANP is formed by amino acids 1    to 98 (proANP₁₋₉₈)

SEQ ID NO: 3 NPMYNAVSNA DLMDFKNLLD HLEEKMPLED EVVPPQVLSE PNEEAGAALSPLPEVPPWTG EVSPAQRDGG ALGRGPWDSS DRSALLKSKL RALLTAPR

-   ProBNP is brain natriuretic peptide prohormone,-   proBNP is processed by cleavage of the N-terminal fragment into the    mature brain natriuretic peptide (BNP). Human proBNP has 108 amino    acids (proBNP₁₋₁₀₈)

SEQ ID NO: 4 HPLGSPGSAS DLETSGLQEQ RNHLQGKLSE LQVEQTSLEP LQESPRPTGVWKSREVATEG IRGHRKMVLY TLRAPRSPKM VQGSGCFGRK MDRISSSSGL GGKVLRRH

-   BNP is brain natriuretic peptide-   Human BNP is formed by amino acids 77 to 108 of the prohormone    (proBNP₇₇₋₁₀₈)

SEQ ID NO: 5 SPKMVQGSGC FGRKMDRISS SSGLGCKVLR RH

-   NT-proBNP is the N-terminal fragment of proBNP-   The N-terminal fragment of human proBNP is formed by amino acids 1    to 76 (proBNP₁₋₇₆)

SEQ ID NO: 6 HPLGSPGSAS DLETSGLQEQ RNHLQGKLSE LQVEQTSLEP LQESPRPTGVWKSREVATEG IRGHRKMVLY TLRAPR

-   proXNP is an agent of the invention which comprises amino acid    sequence derived or originating from both proANP and proBNP-   XNP is an agent of the invention which comprises amino acid sequence    derived or originating from both ANP and BNP-   NT-proXNP is an agent of the invention which comprises amino acid    sequence derived or originating from both NT-proANP and NT-proBNP-   NT-proXNP1 is an agent of the invention formed from proBNP₁₅₋₂₄ and    proANP₈₂₋₉₆-   NT-proXNP2 is an agent of the invention formed from proBNP₁₋₃₇ and    proANP₂₉₋₉₈-   NT-proXNP3 is an agent of the invention formed from proBNP₁₀₋₂₉ and    proANP₂₀₋₈₀-   NT-proXNP4 is an agent of the invention formed from proBNP₁₋₇₆ and    proANP₁₋₉₈-   NT-proXNP5 is an agent of the invention formed from proBNP₁₀₋₂₉ and    proANP₆₀₋₈₀-   ProXNP6 is an agent of the invention formed from proBNP₁₋₁₀₈ or a    subsequence thereof and proANP₁₋₁₂₆ or a subsequence thereof-   XNP7 is an agent of the invention formed from proBNP₇₇₋₉₂ or a    subsequence thereof and proANP₁₁₂₋₁₂₆ or a subsequence thereof.    Variant Polypeptides

Variants of polypeptides are referred to herein. For example, referencesare made to variants of proANP, ANP, NT-proANP, proBNP, BNP andNT-proBNP, in the description of binding substances and agents of theinvention. Reference is also made to variants of the GC-A, GC-B and GC-Creceptor polypeptides.

The term “variant” refers to a polypeptide which has the same essentialcharacter as or a basic biological functionality of the relevantpolypeptide. Thus a variant is typically capable of complementing one ormore activities of that polypeptide. Typically a variant comprises anamino acid sequence which is homologous to all or a part of the sequenceof the polypeptide. In general a (homologous) variant has an amino acidsequence with more than 70% identity, preferably at least 75% or 80% orat least 90% and particularly preferably at least 95%, at least 97% orat least 99% identity with the given sequence, for example over a regionof at least 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 ormore contiguous amino acids. Variants may include allelic variants,species homologues and the deletion, modification or addition of singleamino acids or groups of amino acids within the protein sequence, aslong as the peptide maintains a basic biological functionality of thesubject polypeptide.

An allelic variant will be a variant which will occur naturally, forexample, in a human, and which will function in a substantially similarmanner to the relevant polypeptide. Similarly, a species homologue of aprotein will be the equivalent protein which occurs naturally in anotherspecies and which retains a basic biological function of the givenpolypeptide. Thus, for example, a naturally occurring or nativepolypeptide variant, such as those which may be detected in a biologicalsample, may be an allelic variant or species homologue of another knownpolypeptide.

Allelic variants and species homologues can be obtained, for example, byprobing a library made from cells of the appropriate species using asuitable probe, to obtain clones encoding the allelic or speciesvariants. The clones can be manipulated by conventional techniques togenerate a polypeptide which can be produced by recombinant or synthetictechniques known per se.

Variants may include polypeptides which are longer in length than therelevant polypeptide. A variant may comprise or consist of at least 30,40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, amino acids up to forexample 500, 1000 or 2000 amino acids.

A variant may be a fusion protein.

Variants may include amino acid substitutions, for example from 1, 2 or3 to 10, 20 or 30 (or 10, 20, 30 or 40 to 50, 60 or 70) substitutions.The modified polypeptide generally retains the ability to complement oneor more of the activities of and/or the antigenic activity of thesubject polypeptide. Conservative substitutions may be made, for exampleaccording to the following table. Amino acids in the same block of thesecond column and preferably in the same line in the third column may besubstituted for each other.

ALIPHATIC Non-Polar G A P I L V Polar-uncharged C S T M N QPolar-charged D E K R AROMATIC H F W Y

Shorter polypeptide sequences or fragments are within the scope of theinvention. For example, a peptide of at least 2, 5, 10, 12, 15, 17, 20,25 amino acids or up to 30, 40, 50, 60, 70, 80, 100, 200, 300, 400 or500 amino acids in length (depending on the size of the subjectpolypeptide) is considered to fall within the scope of the invention aslong as it demonstrates a basic biological functionality of the subjectpolypeptide. In particular, but not exclusively, this aspect of theinvention encompasses the situation when the protein is a fragment ofthe complete protein sequence and may represent a binding site foranother molecule or entity, such as a peptide-binding region, or anepitope. Such fragments may or may not retain other functions of thesubject polypeptide.

Variant polypeptides as referred to herein generally retain a basicbiological functionality of the relevant polypeptide. The variant mayretain one or more of the native biological activities or functions ofthe subject polypeptide.

In particular, a variant as referred to herein generally retains one ormore of the binding characteristics of the relevant polypeptide.Alternatively or additionally, the variant may retain an antigenicactivity of the polypeptide.

In one embodiment a variant exhibits at least one of the binding orrecognition properties of the subject polypeptide. In particular avariant may be capable of binding to a product that can bind to thepolypeptide e.g. a ligand, a receptor or an antibody. Thus, for example,a variant of ANP or BNP may be capable of binding to the GC-A receptor.Similarly, a variant of the GC-A or GC-B receptor may bind ANP or BNP.Typically a variant binds the product with an affinity that is at least60%, such as at least 70, 80 or 90%, for example 95, 97 or 99% of theaffinity with which the relevant polypeptide binds to the product.Suitable binding assays are known in the art.

Variants which have a particular activity or binding characteristic ofthe given polypeptide may be identified based on such activities orcharacteristics, for example from a library of polypeptides.

In a further embodiment a variant polypeptide may retain the antigenicproperties of the subject polypeptide. Such a variant may, for example,be capable of generating an immune response in a subject. The immuneresponse may be antibody and/or cell mediated, such as T-cell mediated.Thus a variant may be capable of raising antibodies which are specificfor and bind to the subject polypeptide. A peptide for generating animmune response may be identified by immunisation studies, typically inan animal model. For example, a candidate peptide may be administered toan animal and subsequently the antibody or T-cell response generatedwhich is specific for the peptide may be determined. Antiserum generatedfollowing administration of a peptide to an animal may be evaluated forthe ability to bind the peptide or to bind the subject polypeptide.

A variant which has at least one of the binding characteristics of agiven polypeptide may comprise at least one binding region of thepolypeptide. Such a binding region in general mediates binding of thepolypeptide to another product such as a receptor or an antibody. Thebinding region may be external or internal to the given polypeptide.Thus a variant may comprise a binding site, epitope or antigenicfragment of the relevant polypeptide. Preferably the binding region inthe variant retains the conformation which it has in the relevantpolypeptide. In one aspect the variants are fragments. For example thefragments may be at least 6 amino acids in length, preferably at least10, such as at least 12 or 15 or up to 20, 30 or 40 amino acids. Longerfragments such as up to 60, 90, 100 or 200 amino acids may also be used.Such fragments may not otherwise demonstrate a cellular function oractivity of the subject polypeptide.

ANP and BNP

Cardiac natriuretic peptides ANP and BNP and the N-terminal fragments(NT-proANP and NT-proBNP) of A- and B-type natriuretic peptideprohormones (proANP and proBNP) are released to the circulation when theheart is subjected to pressure or volume overload. Their function is todecrease the load and protect the heart. In spite of the fact that theheart produces two distinct biologically active natriuretic peptides(ANP and BNP), each derived from its own gene and regulated differently,their biological effects are mediated to the target cells by a singlereceptor, GC-A (NPRA) (Drewett et al. 1994). Activation of both the ANPand BNP hormonal systems refers to the up-regulation of both ANP and BNPgenes or production or increase in plasma concentrations, whereasinactivation of both ANP and BNP systems refers to the down-regulationof both ANP and BNP genes or production or decrease in plasmaconcentration.

In cardiac pressure and volume overload, ANP gene expression and thecirculating levels of ANP and NT-proANP are primarily induced byincreased preload of the heart, whereas BNP gene expression andcirculating levels of BNP and NT-proBNP are primarily sensitive to anincrease of afterload (Yoshimura et al. 1993; Yasue et al. 1994).Moreover, ANP and BNP genes are regulated differentially in thedifferent chambers of the heart (Dzimiri et al. 2002). Elevated plasmaANP or NT-proANP levels are associated with atrial overload e.g.tachycardia, whereas BNP and NT-proBNP are, better markers ofventricular overload e.g. aortic stenosis. Markedly elevated circulatinglevels of both ANP and BNP (and NT-proANP and NT-proBNP) suggestcombined atrial and ventricular overload, as in dilated cardiomyopathy.Thus, in physiological and pathophysiological situations the informationmediated by ANP and BNP converges in the target cell membrane to cause acommon intracellular signalling cascade. As already mentioned existingassays measure one of the analytes at a time (ANP, BNP, NT-proANP orNT-proBNP). Because a major strength of the natriuretic peptides in thediagnosis of cardiac diseases lies in the high negative predictivevalue, the combination assay of ANP and BNP (or NT-proANP and NT-proBNP)in the present invention will add value over that provided by assayingany of the analytes alone. Sequential assay of ANP and BNP (or NT-proANPand NT-proBNP) would be unnecessarily complex, including doubled effortfor quality control, and not as cost-effective.

The present invention provides novel diagnostic methods and use thereofwhich, by mimicking the physiological regulatory system working in thebody, can combine the information obtained from the activation orinactivation, respectively, of the ANP and the BNP hormonal systems by asimple means of simultaneous measurement of a proportionally cumulativeconcentration of peptides derived from both A- and B-type natriureticpeptide prohormones.

Test Methods

The invention provides a new and sensitive method suitable fordiagnosing and assessing cardiac conditions such as heart failure, whichdetermines activation or inactivation of the ANP and BNP hormonalsystems. The method of the invention focuses on detecting or monitoringthe combined levels of proANP, proBNP and fragments thereof in asuitable biological sample. According to the method, peptides derivedfrom or originating from both proANP and proBNP may be assayed at thesame time in a given sample. In one embodiment the method may be usedfor screening for diagnostic purposes or for monitoring treatment.

Peptides derived from proANP and proBNP include NT-proANP, ANP,NT-proBNP, BNP as well as proANP and proBNP. The sample may compriseother peptides also derived from the prohormones eg by proteolysis. Forexample, other peptides may include proANP₁₋₃₀ proANP₃₁₋₆₇ orproANP₇₉₋₉₈. Thus in one embodiment, any combination of proANP,NT-proANP, ANP, proBNP, NT-proBNP and BNP, and optionally other derivedpeptides, may be assayed in the method. In one embodiment the sample maycontain proANP-derived peptides without proBNP-derived peptides or viceversa.

Since the peptides are detected simultaneously, only a single reading orresult is required. The method is suitable for assessing the risk of anddetecting cardiac impairment such as heart failure, and for evaluatingtreatments for heart failure. As such it is more sensitive, cheaper andsimpler to perform than prior art methods.

The peptides assayed in the invention are present at normal referencelevels in the general population. Activation of the ANP and BNP systemsmay be considered as occurring when the combined peptide level isgreater than this normal reference level. Therefore in any particularassay format, if the result indicates a qualitatively or quantitativelyhigher peptide level than the reference level, activation of the systemsis implied. For example, an assay may be calibrated eg using an agent ofthe invention, so that a particular reading in the assay is known torepresent the normal peptide level. Or the assay may be such that anormal or reference level of peptide will produce a negligible orinsignificant result.

Inactivation of the ANP and BNP systems, for example after heart failureperhaps in response to medical treatment, occurs when the combinedpeptide level falls from the elevated level associated with the earliercardiac incident. By performing serial assays it will be possible todetect a qualitative or quantitative decrease in peptide levels. It willbe possible to determine also the rate of decrease and so to assess theeffectiveness of a given treatment.

The present methods are capable of simultaneously detecting bothproANP-derived and proBNP-derived peptides. The actual change inindividual peptide levels may be for example A+ and B+, A+ and B−, A−and B+, or A− and B− (where A represents levels of proANP-derivedpeptides and B represents levels of proBNP-derived peptides).

The present assay methods may be qualitative or quantitative. Forexample, a quantitative assay is possible when an agent of the inventionis used as a competing antigen in a competition assay.

In general, the present method comprises contacting a sample with afirst binding substance which is able to bind both proANP- andproBNP-derived peptides under conditions which will allow such bindingto occur. Any binding complexes formed between the first bindingsubstance and such peptides are then detected. Suitable detection meansare known in the art and are described in more detail below.

The peptides to be detected are as described above. In one aspect theyare naturally occurring peptides. The peptides provide an indicator ofactivation or inactivation of the ANP and BNP systems. The first bindingsubstance is as defined herein.

In one embodiment the first binding substance is a bi- or oligo-specificbinding substance as defined herein. Such a binding substance is able tobind to both proANP- and proBNP-derived peptides. In one embodiment thisfirst binding substance is used in the assay when an agent of theinvention is not used in the assay, for example as a competing antigenin a competitive binding assay. Binding complexes between the firstbinding substance and the peptides in the sample may be detected andactivation or inactivation determined as above.

In one aspect, the present method additionally comprises contacting asample with an agent of the invention as described herein (XNP, proXNP,or NT-proXNP). The agent comprises peptides derived from or originatingfrom both proANP and proBNP and is able to bind to the first bindingsubstance. Such an agent may be used as a standard to calibrate thepresent assays. The agent may be used as a competing antigen in acompetition assay. Peptide levels in a given sample may thus beexpressed in terms of agent concentration. Suitable assay formats,detection and quantifying means are known in the art and are describedin more detail below.

The methods of the invention are generally applied to a sample,typically a biological sample. Typically the sample is one which isknown or suspected of being a body sample from an individual, such as ahuman. A sample may be one taken from an individual or patient. Thesample may comprise a body fluid, e.g. blood, serum, plasma,cerebrospinal fluid, urine, saliva or other biological fluid in whichpeptides derived from A- and B-type natriuretic peptide prohormonesmight be present. The sample may be a human sample. In one embodimentthe sample is obtainable from an individual or patient using a standardor routine procedure. The sample may therefore be such that the assaycan be used for diagnostic screening or therapeutic monitoring orassessment. In one aspect the sample is obtainable from a livingindividual or subject.

A sample may be processed before it is used in the method. For example,it may be diluted, typically in water, saline or saline containing abuffer (any of these diluents may additionally comprise detergent).

Generally, the present method is carried out in aqueous solution.However, in particular embodiments (some of which are discussed below),the first binding substance, or the agent may be immobilised in a solidsupport. Typically such a support is the surface of the container inwhich the method is being carried out, such as the surface of a well ofa microtitre plate. In other embodiments the support may be a sheet(e.g. a nitrocellulose or nylon sheet) or a bead (e.g. sepharose orlatex).

In one embodiment the solid support is a particle, dipstick ormicrotitre plate. An ELISA plate may be used.

The first binding substance or the agent may be labelled with adetectable label. Examples of suitable labels have been describedherein.

In principle, any suitable assay technique may be employed in thepresent invention. For example, suitable methods include immunoassaymethods, both competitive and non-competitive, antibody binding methodsemploying either unlabelled or labelled antigens or their analogues(immunoassays), or labelled or unlabelled binding substances recognisingtheir antigens or analogues (immunometric assays and receptor bindingassays), respectively. Sandwich assays may be used.

Immunoassay methods which may be used include europium fluorescenceimmunoassays (FIA), enzymelinked immunoabsorbent assays (ELISA),radioimmunoassay (RIA), immunoradiometric assay, enzyme immunoassay,immunoenzymometric assay, time-resolved fluoroimmunoassay,immunofluorometric assay, chemiluminescence immunoassay (CLIA), anodicor cathodic electrochemiluminescence immunoassays, various dry-chemistrytest strip assays, particle based immunoassays, direct labellessimmunoassays, such as assays based on surface plasmon resonance, surfaceacoustic waves and surface-enhanced Raman spectroscopy, homogeneicimmunoassays such as proximity assays with two different labels, chiptechnology, array technology, particle enhanced immunoassays and otherparticle immunoassays, both single and dual size labelled or unlabelledparticles. Latex and gold, in different forms, can be mentioned asexamples of particles to be used. Turbidometric and nephelometricdeterminations are also possible assay formats.

Chromatographic membrane technology can also be used as a format toimplement the present invention. The chromatographic membrane testcomprises both a lateral and flow-through test. The used reagents areeither permanently or non-permanently immobilised onto the membranewhere they have a very distinct role in the different zones of the testi.e. one or multiple zone(s) for reagent(s), test(s), control(s) etc.The reagents immobilised can be binding substance, the agent of theinvention, anti-binding substance antibody, anti-analyte antibody,anti-agent antibody or a label.

Binding complexes of the first binding substance with peptides in thesample or with the agent may be detected using a second bindingsubstance. For example, the second binding substance may be an antibodywhich itself bears a detectable label such as those listed above. Thesecond binding substance may be a substance that causes precipitation orotherwise immobilises and separates the first binding substancecomplexes.

Particular embodiments of the present method will now be described inmore detail:

-   (a) One embodiment uses labelled proXNP agent as antigen, together    with antibody as first binding substance recognising both peptides    derived from both A- and B-type natriuretic peptide prohormones and    the agent. In such methods a known constant amount of labelled agent    is added to the sample containing an unknown amount of unlabelled    antigen, i.e. peptide analyte to be measured. Both the labelled and    the unlabelled antigen bind to the first binding substance, for    example in a competitive manner and measurement of the amount of the    bound labelled agent, when compared to the known amount agent added,    can be used to determine how much unlabelled antigen is present in    the sample thus reflecting the activation or the inactivation of    both the ANP and the BNP systems as a proportionally cumulative    measure of peptides derived from both A- and B-type natriuretic    peptide prohormones.-   (b) Another preferred type of method uses a labelled first binding    substance. In such methods, the complex of labelled first binding    substance and peptides derived from both A- and B-type natriuretic    peptide prohormones is assayed giving a proportionally cumulative    measure of the amount of peptides derived from both A- and B-type    natriuretic peptide prohormones in the sample. A specific case is    the one where the first binding substance can be the natriuretic    receptor GC-A or a fragment or extension thereof, a bi-,    oligospecific or bifunctional antibody recognising peptides derived    from both A- and B-type natriuretic peptide prohormones and agent.-   (c) An additional type of method relies on the use of a labelled    antibody to binding substance, which antibody may be produced in a    different animal species than the used first or secondary antibody    in case of binding substance comprising an antibody.-   (d) A further method comprises (i) contacting the sample with an    agent and a first binding substance, comprising labelled first    binding substance or comprising labelled agent; and (ii) detecting    and/or quantitatively determining the binding of the labelled first    binding substance to the unlabelled agent or labelled agent to the    binding substance that recognises peptides derived from both A- and    B-type natriuretic peptide prohormones and agent.-   (e) One method comprises contacting a sample, which sample is known    or suspected to contain peptides derived from both A- and B-type    natriuretic peptide prohormones with (in any order): (i) a first    binding substance which recognises both peptides derived from both    A- and B-type natriuretic peptide prohormones and an agent of the    invention; and (ii) a known amount of the labelled agent, which acts    as an antigen, such that the label is bound to the binding substance    in an amount which depends on the amount of unlabelled peptides    derived from both A- and B-type natriuretic peptide prohormones    present in the sample; and assaying the amount of the bound and/or    unbound label as a proportionally cumulative measure of unlabelled    level of peptides derived from both A- and B-type natriuretic    peptide prohormones in the sample.-   (f) A conventional immunoassay method (e.g. radioimmunoassay) may    comprise:    -   (i) immobilising on a solid support unlabelled first binding        substance recognising and binding peptides derived from both A-        and B-type natriuretic peptide prohormones and agent of the        invention;    -   (ii) adding a sample containing or suspected of containing the        target native peptides derived from both A- and B-type        natriuretic peptide prohormones together with a fixed amount of        labelled agent, such that the peptides derived from both A- and        B-type natriuretic peptide prohormones and the labelled agent        are free to compete for binding to the immobilised binding        substance;    -   (iii) separating out the immobilised (bound) material from the        non-immobilised (unbound) material;    -   (iv) determining the amount of binding substance-bound labelled        agent; and    -   (v) comparing the amounts of bound or unbound labelled agent in        assay mixtures of test samples with the signal obtained using        calibrators with known concentration of agent in order to        determine the proportionally cumulative concentration of        peptides derived from both A- and B-type natriuretic peptide        prohormones in the sample being assayed.-   (g) Alternatively, method (f) can be performed in solution, wherein,    a second binding substance can be used to either precipitate or    otherwise immobilise and separate the first binding    substance-antigen complexes. A typical example of this comprises:    -   (i) contacting a sample containing or suspected of containing        the peptides derived from A- and B-type natriuretic peptide        prohormones to be detected with a first binding substance        recognising peptides derived from both A- and B-type natriuretic        peptide prohormones or binding thereto according to the        invention in the presence of a fixed amount of a labelled agent        of the invention;    -   (ii) contacting the resulting mixture with an immobilised        secondary binding substance which binds to the first binding        substance;    -   (iii) separating out the immobilised material from the        non-immobilised material; and    -   (iv) comparing amounts of the labelled agent in the immobilised        or non-immobilised material with the amounts obtained using        calibrators with known concentration of novel agent to determine        the proportionally cumulative concentration of peptides derived        from both A- and B-type natriuretic peptide prohormones in the        sample being assayed.-   (h) An immunometric assay employing the use of immobilised    unlabelled agent is also envisioned, a typical example of which    comprises:    -   (i) immobilising on a solid support unlabelled agent of the        invention;    -   (ii) adding a sample containing or suspected of containing the        target peptides derived from A- and B-type natriuretic peptide        prohormones together with a fixed amount of labelled binding        substance which recognises peptides derived from both A- and        B-type natriuretic peptide prohormones according to the        invention, in such a way that peptides derived from both A- and        B-type natriuretic peptide prohormones in the sample are free to        compete with the immobilised agent of the invention for the        labelled binding substance.    -   (iii) separating out the labelled first binding substance        recognising peptides derived from both A- and B-type natriuretic        peptide prohormones that is not bound to the immobilised agent        of the invention;    -   (iv) determining the amount of labelled binding substance bound        to the immobilised agent of the invention; and    -   (v) comparing the amounts of immobilised or non-immobilised        labelled binding substance in the assay mixtures of test samples        with the activity obtained using calibrators with known        concentration of agent of the invention, in order to determine        the proportionally cumulative concentration of peptides derived        from both A- and B-type natriuretic peptide prohormones in the        sample being assayed.

Thus the invention provides methods for determination of theproportionally cumulative concentration of peptides derived from both A-and B-type natriuretic peptide prohormones in a sample, showing eitheran activation or inactivation of both the ANP and BNP systems.

The First Binding Substance

According to the present method, peptides derived from both proANP andproBNP may be assayed at the same time in a given sample. As abovepeptides derived from proANP and proBNP include NT-proANP, ANP,NT-proBNP, BNP as well as proANP and proBNP. A sample may comprise otherpeptides also derived from the prohormones eg by proteolysis. Forexample, other peptides may include proANP₁₋₃₀, proANP₃₁₋₆₇ orproANP₇₉₋₉₈. Thus in one embodiment, any combination of proANP,NT-proANP, ANP, proBNP, NT-proBNP and BNP, and optionally other derivedpeptides, may be assayed in the method.

The present assays use a first binding substance which recognises orbinds to peptides derived from both A- and B-type natriuretic peptideprohormones, such as those peptides described above. Thus the substanceis able to bind to both proANP and proBNP or to variants, includingfragments of both prohormones. In one embodiment the first bindingsubstance may not bind both sets of peptides with equal affinity. Thebinding substance may bind to naturally occurring proANP, ANP orNT-proANP and/or to naturally occurring proBNP, BNP, or NT-proBNP. Forexample it may bind to SEQ ID Nos 1, 2 or 3 and SEQ ID Nos 4, 5 or 6, orto allelic variants or species homologues thereof. Alternatively oradditionally, the substance may bind to one or more fragments of any ofthe above sequences, for example fragments which include an epitope,antigenic fragment, or a binding site. Such fragments are discussed inmore detail herein.

In one aspect the first binding substance is able to bind to both:

-   (a) (i) proANP (SEQ ID NO. 1), ANP (SEQ ID NO. 2) or NT-proANP (SEQ    ID NO. 3);    -   (ii) a homologous variant of (i); or    -   (iii) a fragment of (i) or (ii);-   and-   (b) (i) pro-BNP (SEQ ID NO. 4), BNP (SEQ ID NO. 5) or NT-proBNP (SEQ    ID NO. 6);    -   (ii) a homologous variant of (i); or    -   (iii) a fragment of (i) or (ii).

Variants and fragments are as defined herein.

In one embodiment, the homologous variant (ii) has at least 70% identityto (i), and/or the fragment (iii) is at least 6 amino acids in length.In one aspect the homologous variant (ii) is a species homologue or anallelic variant of (i).

In one embodiment a binding substance according to the invention is ableto bind to a peptide comprising or consisting of amino acids 7 to 23 ofANP and/or amino acids 10 to 26 of BNP or variants thereof. Thesepeptide regions form a conserved ring structure in the native molecules.

A binding substance may bind to an epitope of proANP and/or proBNP. Forexample, suitable epitopes include amino acids 5-13, 1-10, 15-25 and27-32 of BNP and amino acids 65-76 and 1-13 of NT-proBNP or variantsthereof

In one embodiment, a binding substance is able to bind to one or morepeptides selected from proBNP₁₋₃₇, proBNP₁₅₋₂₄, proBNP₁₀₋₂₉,proBNP₇₇₋₉₂, proBNP₁₋₁₀₈, proANP₂₉₋₉₈, proANP₈₂₋₉₆, proANP₂₀₋₈₀,proANP₆₀₋₈₀, proANP₁₋₁₂₆ and proANP₁₁₂₋₁₂₆ or variants thereof. Forexample, a binding substance may be able to bind to both proBNP₁₅₋₂₄ andproANP₈₂₋₉₆, to both proBNP₁₋₃₇ and proANP₂₉₋₉₈, to both proBNP₁₀₋₂₉ andproANP₂₀₋₈₀, to both proBNP₁₀₋₂₉ and proANP₆₀₋₈₀, to both proBNP₁₋₁₀₈and proANP₁₋₁₂₆ or to both proBNP₇₇₋₉₂ and proANP₁₁₂₋₁₂₆. For example, abinding substance according to the invention may bind to NT-proXNP1 (SEQID NO:13), NT-proXNP2 (SEQ ID NO:14), NT-proXNP3 (SEQ ID NO:15)NT-proXNP4 (SEQ ID NO:17), NT-proXNP5 (SEQ ID NO: 18), proXNP6 (SEQ IDNO: 19) or XNP7 (SEQ ID NO: 20).

In those embodiments of the present invention which use a binding agentof the invention, the first binding substance is also able to bind tothe binding agent.

Suitable binding assays for determining binding are known in the art. Ingeneral a first binding substance is able to bind a given peptide to anextent that it can be used in a binding and detection assay such asthose described herein. For example, a suitable binding substance maybind with at least 50, 60, 70, 80, 90, 95 or 100% of the bindingaffinity of a specific antibody to the peptide, or of the natriureticreceptor GC-A to the peptide, e.g. of receptor GC-A to ANP or BNP.

The first binding substance as used herein may be a single substance ora mixture of substances. A suitable binding substance may be forexample, a receptor or antibody, or fragments or derivatives thereof,with bi- or oligo-specific properties, or a mixture thereof.

In one aspect a mixture of binding substances is used as a first bindingsubstance in embodiments where an agent of the invention is also used. Amixture may comprise monospecific, bispecific and/or oligospecificbinding substances. Any suitable composition of binding substances maybe used that allows detection of proANP- and proBNP-derived peptidesaccording to the present methods. pro-ANP-derived peptide specificbinding substances and proBNP-derived peptide specific bindingsubstances may be present in any suitable proportions. For example theymay be present in equal amounts or binding capacities. Alternatively,each may be present at, for example, 2×, 3×, 4×, 5× up to 10× the amountor binding capacity of the other. In one embodiment, a 1:1 mixture ofproANP-derived peptide-specific binding substance eg antibody andproBNP-derived peptide specific binding substance eg antibody may beused in an assay.

In embodiments where the assay does not include agent of the inventionit is preferred that the first binding substance is a single bi- oroligo-specific binding substance. Thus a first binding substance for usein such embodiments may be a single substance that is bi- oroligo-functional in binding. That single substance has the bindingspecificity of the first binding substance as set out above. It may forexample, have two or more ligand binding sites, or two or more ligandsmay bind to one site in the substance with the same or differentaffinities. For example such a substance may comprise a receptor orantibody or fragments of either.

(a) Receptors

An example of a suitable receptor is natriuretic receptor GC-A. Asequence of human GC-A may be found under accession no. P16066 (SEQ IDNO:33). The receptor or a fragment, extension or derivative thereof canbe produced by methods known to those skilled in the art (Misono et al.,1999). Briefly, the extracellular ligand binding domain of the receptor(SEQ ID NO:34) may be produced by cloning the DNA sequence encoding theamino acid sequence required for binding both human ANP and BNP, into asuitable prokaryotic or eukaryotic expression vector, transfecting thevector into an appropriate host cell, growing the host cell in asuitable culture medium, and harvesting the recombinant protein. Thereceptor-derived sequence may be released by enzyme digestion, purifiedwith affinity chromatography and reverse-phase HPLC and identified bypeptide mapping and sequencing.

The GC-B receptor (accession no. P20594, SEQ ID NO:35) or ANPrecC orclearance receptor (accession no. P17342, SEQ ID NO:36) may also act asa binding substance.

Thus in one embodiment the first binding substance may comprise:

-   -   (a) natriuretic receptor GC-A (SEQ ID NO: 33), GC-B (SEQ ID        NO: 35) or GC-C (SEQ ID NO: 36);    -   (b) a homologous variant of (a); or    -   (c) a fragment of (a) or (b).

In one aspect the first binding substance comprises an extracellularbinding domain of the natriuretic receptor GC-A (SEQ ID NO: 34) or ahomologous variant of fragment thereof.

An extension or derivative of any of the above binding substances may beused. Thus the structure of the molecule may be modified, for example byadding a handle to facilitate attachment to a solid support, while stillretaining the binding ability or properties of the molecule.

(b) Antibodies

The first binding substance may comprise an antibody or a fragment orderivative of an antibody. Thus in one aspect the present inventionrelates to antibody with the binding specificity set out above.

Antibodies may be raised against specific epitopes of the given peptidesequences. An antibody, or other compound, “specifically binds” to apolypeptide when it binds with preferential or high affinity to theprotein or proteins for which it is specific but does substantially notbind or binds with only low affinity to other polypeptides. A variety ofprotocols for competitive binding or immunometric assays to determinethe specific binding capability of an antibody are well known in the art(see for example Maddox et al, J. Exp. Med. 158, 1211-1226, 1993). Suchimmunoassays typically involve the formation of complexes betweenspecific protein and antibody and the measurement of complex formation.

An antibody according to the invention may comprise either a wholeantibody or a fragment thereof and has the binding specificity set outabove. Fragments include Fv, F(ab′) and F(ab′)₂ fragments, as well assingle chain antibodies. A whole antibody is typically an antibody whichis produced by any of the methods of producing an antibody which arediscussed herein. Typically the antibody is a mammalian antibody, suchas a primate, human, rodent (e.g. mouse or rat), rabbit, ovine, porcine,equine, goat or camel antibody. The antibody can be any class or isotypeof antibody, for example IgM, but is preferably IgG.

The antibody may be a bispecific antibody which is able to bind to twodifferent antigens, or an oligospecific antibody which is able to bindto more than two different antigens. The antibody may comprise apolyclonal, monoclonal, oligoclonal, bifunctional or crossreactingpolyclonal antibody as explained above.

A fragment of whole antibody that can be used in the method comprises anantigen binding site, e.g. F(ab′) or F(ab)₂ fragments. Such fragments orantibodies may be used to form antibody derivatives. For example thewhole antibody or fragment may be associated with other moieties, suchas linkers which may be used to join together two or more fragments orantibodies. Such linkers may be chemical linkers or can be present inthe form of a fusion protein with the fragment or whole antibody. Thelinkers may thus be used to join together whole antibodies or fragmentswhich have the same or different binding specificities, e.g. that canbind the same or different polymorphisms. The antibody may be a“diabody” formed by joining two variable domains back to back. In oneembodiment the antibody is a chimeric antibody comprising sequence fromdifferent natural antibodies, for example a humanised antibody.Bifunctional antibodies may be made by chemical combination of fragmentswith desired characteristics.

Antibodies of the invention can be produced by any suitable method. Forexample, antibodies, fragments or derivatives thereof may be produced byselecting immunogens to raise antibodies, chemically coupling antibodiesor antibody fragments, somatic fusion of monoclonalhybridomas/splenocytes or recombinant techniques. Phage displaytechniques may be used in antibody production.

Means for preparing and characterising antibodies are well known in theart, see for example Harlow and Lane (1988) “Antibodies: A LaboratoryManual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.For example, an antibody may be produced by raising antibody in a hostanimal against the whole polypeptide or a fragment thereof, for examplean antigenic epitope thereof, or “immunogen”. The fragment may be any ofthe fragments mentioned herein (typically at least 6 or at least 10 or15 amino acids long). An agent of the invention may be used to raiseantibody using known techniques.

A method for producing a polyclonal antibody comprises immunising asuitable host animal, for example an experimental animal, with theimmunogen and isolating immunoglobulins from the animal's serum. Theanimal may therefore be inoculated with the immunogen, bloodsubsequently removed from the animal and the IgG fraction purified.

Polyclonal and monoclonal antibodies are produced by immunising asuitable host animal (e.g. rabbit, sheep, goat, swine, chicken, guineapig, rat or mouse) with an immunogen. For example, the immunogen maycomprise an agent according to the present invention. In one embodimentone or more boosters of immunogen are administered to the animal. Forexample, 1, 2, 3, 4 or more boosters may be used. Methods of producingpolyclonal or monoclonal antibodies are well-known for those skilled inthe art and any of these methods may be used to prepare antibodies. Ifdesired, the immunogen may be administered as a conjugate, in which theimmunogen is coupled, for example via a side chain of the amino acidresidues to a suitable carrier. The carrier molecule is typically aphysiologically acceptable carrier.

After the experimental animal has produced polyclonal antibodies theserum may be used diluted or the desired immunoglobulins may be isolatedfrom the serum. In one embodiment the serum may be diluted before use.Suitable dilutions may include for example, from 1:1000 to 1:500,000,for example from 1:20,000 to 1:80,000, 1:10,000 to 1:15,000 or from1:50,000 to 1:60,000. In one embodiment the concentration of antibodyused in an assay of the invention is the same as the concentration ofantibody in such a dilution of serum. The obtained antibody may beisolated and, if desired, purified, for example to a purity of 70%-100%.Typically the animal is inoculated with immunogen, the blood is removedand the IgG fraction is purified.

The methods for producing monoclonal antibodies are also well-known forthose skilled in the art (Köhler & Millstein 1975 Nature 256, 495-497).Such methods generally comprise immortalising cells which produce theclaimed antibody. Hybridoma cells producing monoclonal antibodies, areproduced by fusing spleen cells from an immunised animal with tumourcells. The resulting hybridoma cells are immortalised and the cellsproduce the desired antibody. The immortalised cell producing thedesired antibody may be selected by a conventional procedure. Thehybridomas may be grown in culture or injected intraperitoneally, forformation of ascites fluid, or into the bloodstream of an allogeneic orimmunocompromised host.

Human antibodies may be produced by in vitro immunisation of humanlymphocytes, followed by transformation of the lymphocytes withEpstein-Barr virus. Monoclonal antibodies may also be produced byrecombinant DNA technology as described by Skerra and Plückthun (1988).It is also possible to use any derivates, as for example F(ab′), andF(ab′)₂ fragments of both monoclonal and polyclonal antibodies preparedby proteolytic reaction of papain and pepsin, respectively, onsubstantially intact antibodies by methods well known for a personskilled in the art.

Thus the antibodies provided by the invention (and those which are usedin the method of the invention) may be made by culturing a cell thatexpresses the antibody and optionally purifying the antibody from thecell.

The cell used in the process may be one which is obtainable byadministering a peptide comprising any of the relevant antigenicpeptides to a mammal, extracting B cells from the mammal and selecting acell from these based on the ability to express an antibody which bindsthe antigens. Optionally the B cells are immortalised after extractionor selection, for example by fusing them with an immortal cell (to forma hybridoma) or by infection with EBV virus.

Cells that express the antibody comprise a polynucleotide that iscapable of expressing the antibody, a polynucleotide that encodes theantibody.

Another type of cell which can be used to make the antibody is one whichis recombinant for a polynucleotide which expresses the antibody. Such acell may be prokaryotic or eukaryotic (such as from any of the mammalsmentioned herein).

Antibody may be immobilised on a solid support. Typically the solidsupport is the surface of the container in which the method is beingcarried out, such as the surface of a microtitre plate. In otherembodiments the support may be a particle, a sheet (e.g. anitrocellulose or nylon sheet) or a bead (e.g. Sepharose or latex).Antibody may be present on an ELISA plate or in a dipstick.

Antibodies of the invention are for example useful in purification,isolation or screening methods involving immunoprecipitation techniques.

The invention also includes a dipstick which can be used to carry outthe method of the invention. The dipstick comprises a porous materialcapable of chromatographically, transporting a liquid and one or more ofthe antibodies mentioned herein. When the dipstick is contacted with thesample it draws up liquid from the sample towards a detection region onthe dipstick. Peptides in the sample which derive from proANP or proBNPare detected by their binding to the detection region.

In one embodiment the liquid is drawn through a region in the dipstickcontaining the antibodies of the invention. These antibodies bind to therelevant peptides forming an antibody/peptide complex. This complex isdrawn towards the detection region which contains an agent (immobilisedon the dipstick) that binds and thus immobilises the complex in thedetection region. This agent is typically a specific binding agent (e.gan antibody) that binds either the antibody or the peptide of thecomplex. The antibody/peptide complex is typically detected in thedetection region by the use of a label which is attached to the specificantibody.

In another embodiment protein in the sample is labelled before it isdrawn up the dipstick. The labelled protein is then drawn up thedipstick (which has been contacted with sample) and is detected bybinding the polymorphism specific antibody (which is bound to thedetection region).

Typically the label used in the dipstick systems described above is avisually detectable label which becomes visually detectable (i.e. can beseen with the human eye) when enough antibody/protein complex becomesimmobilised in the detection region. A suitable label is a gold (orother colloidal metal) particle or a fluorophore (e.g. fluoroscein).

The dipstick may comprise a denaturing agent that causes denaturation ofthe protein which is drawn up the dipstick. In one embodiment the sampleis exposed to denaturing conditions (e.g. contacted with a denaturingagent) before it is contacted with the dipstick.

Agents of the Invention (proXNP, NT-proXNP, XNP)

(a) Peptide Agents

In one embodiment the present test method utilises an agent (proXNP, XNPor NT-proXNP) which comprises amino acid sequence derived or originatingfrom both proANP and proBNP. The agent mimics proANP- and proBNP-derivedpeptides in the sample to be tested in particular, naturally occurringpeptides. The agent for use in the present method is also recognised orbound by the first binding substance to be used in the method. Thus theagent can compete with the peptides in a sample for binding to the firstbinding substance in the assays of the invention. An agent may also beused as a calibrator or standard in an assay. Thus the agents areparticularly useful for quantifying peptides derived from proANP andproBNP in a sample. For example, in the present assay methods, themeasure of peptides in a sample may be expressed as a concentration ofagent. Furthermore the agent may be used as an immunogen to produceantibody suitable for use as a first binding substance, according to themethods set out above.

The agent may comprise or consist essentially of a peptide, polypeptideor protein. For example, an agent may comprise or consist of a fusionprotein. An agent according to the invention generally comprisessequence characteristic of proANP and sequence characteristic of proBNP.Thus an agent typically includes at least one peptide sequence derivedfrom proANP and at least one peptide sequence derived from proBNP.

In particular an agent may comprise both:

-   -   (a) (i) SEQ ID Nos 1, 2 or 3;        -   (ii) an homologous variant of (i); or        -   (iii) a fragment of (i) or (ii);

-   and    -   (b) (i) SEQ ID Nos 4, 5 or 6;        -   (ii) an homologous variant of (i); or        -   (iii) a fragment of (i) or (ii).

In one embodiment the agent comprises both:

-   -   (a) (i) SEQ ID Nos 1, 2 or 3;        -   (ii) a homologous sequence having at least 70% identity to            (i); or        -   (iii) a fragment of (i) or (ii) which is at least 6 amino            acids in length;

-   and    -   (b) (i) SEQ ID Nos 4, 5 or 6;        -   (ii) a homologous sequence having at least 70% identity to            (i); or        -   (iii) a fragment of (i) or (ii) which is at least 6 amino            acids in length.

In one aspect an agent comprises amino acids 7 to 23 of ANP and/or aminoacids 10 to 26 of BNP, or variants thereof. These peptides form aconserved ring structure in nature ANP and BNP, which may be conservedin the agent. In one embodiment, an agent comprises an epitope of proANPand/or proBNP. For example, suitable epitopes include amino acids 5 to13, 1 to 10, 15 to 25 or 27 to 32 of BNP and amino acids 65 to 76 or 1to 13 of NT-proBNP. In one embodiment an agent includes peptide sequencederived (according to the above) from both NT-proANP and NT-proBNP (suchan agent is referred to as NT-proXNP), or from both ANP and BNP (theagent being then referred to as XNP).

In one embodiment the agent may comprise peptide sequence selected fromone or more of proBNP₁₋₃₇, proBNP₁₅₋₂₄, proBNP₁₀₋₂₉, proBNP₇₇₋₉₂,proBNP₁₋₁₀₈, proANP₂₉₋₉₈, proANP₈₂₋₉₆, proANP₂₀₋₈₀, proANP₆₀₋₈₀.proANP₁₁₂₋₁₂₆ or variants thereof. In one aspect an agent comprises atleast one proBNP and at least one proANP sequence selected from thislist. Suitable combinations are proBNP₁₅₋₂₄ and proANP₈₂₋₉₆, proBNP₁₋₃₇and proANP₂₉₋₉₈, proBNP₁₀₋₂₉ and proANP₂₀₋₈₀, proBNP₁₀₋₂₉ andproANP₆₀₋₈₀, proBNP₁₋₁₀₈ and proANP₁₋₁₂₆ or proBNP₇₇₋₉₂ andproANP₁₁₂₋₁₂₆. Thus an agent according to the invention may comprise orconsist of NT-proXNP1 (SEQ ID NO:13), NT-proXNP2 (SEQ ID NO:14),NT-proXNP3 (SEQ ID NO:15), NT-proXNP4 (SEQ ID NO:17), NT-proXNP5 (SEQ IDNO: 18), proXNP6 (SEQ ID NO: 19) or XNP7 (SEQ ID NO: 20).

In addition to the peptide sequences derived from proANP and proBNP theagent may include linker, connector or adduct amino acid sequence ofvariable length or composition. Suitable linkers are known in the art.The structure of the linker may be such as to allow attachment of one ormore labels (eg fluorescent groups or enzymes) to the linker. Forexample, suitable spacers and adducts include Gly-Lys-Tyr-Gly (GKYG)(SEQ ID NO: 16), Ser-Arg, Gly-Ser or a single amino acid such as Tyr orCys. The Tyr residue permits radioiodination and the Lys or Cys residueallows attachment of labels requiring an amino group or sulphydrylgroup.

An agent may be immobilised on a solid support, such as those describedfor antibodies.

An agent of the invention may comprise chemically modified amino acidsequence, e.g. post-translationally modified. For example, it may beglycosylated or comprise modified amino acid residues. It may bemodified by the addition of histidine residues to assist purification.It may be desirable to produce peptide or protein in a form suitable forattachment to a solid support. Protein or peptide may thus be modifiedto enhance its binding to a solid support for example by the addition ofa cysteine residue.

(b) Polynucleotides Encoding Agents

The invention also relates to polynucleotides which encode an agentaccording to the invention or the peptide part of an agent according tothe invention. Such polynucleotides comprise sequence which encodes theagent peptides as defined above and/or sequence which is complementaryto the coding sequence.

In particular a polynucleotide of the invention may comprise both:

-   (a) (i) SEQ ID NOs 7, 8 or 9;    -   (ii) a sequence complementary to (i);    -   (iii) a sequence which hybridises under stringent conditions        to (i) or (ii);    -   (iv) a sequence which is degenerate as a result of the genetic        code to (i), (ii) or (iii);    -   (v) a sequence having at least 70% identity to any of the        sequences in (i) to (iv); or    -   (vi) a fragment of any of the sequences in (i) to (v);-   and-   (b) (i) SEQ ID NOs 10, 11 or 12;    -   (ii) a sequence complementary to (i);    -   (iii) a sequence which hybridises under stringent conditions        to (i) or (ii);    -   (iv) a sequence which is degenerate as a result of the genetic        code to (i), (ii) or (iii);    -   (v) a sequence having at least 70% identity to any of the        sequences in (i) to (iv); or    -   (vi) a fragment of any of the sequences in (i) to (v).

A polynucleotide may also comprise nucleotide sequence encoding linkeror spacer amino acid sequence in the agent. A polynucleotide of theinvention typically comprises 1000 base pairs or less, for example 500base pairs or less. A polynucleotide may comprise up to 200, 300, 400,500, 600, 700, 800 or 900 base pairs. For example, a polynucleotide maycomprise up to 50, 100, 150 or 175 nucleotides.

Typically the polynucleotide is DNA. However, the invention may compriseRNA polynucleotides. The polynucleotides may be single or doublestranded, and may include within them synthetic or modified nucleotides.

A polynucleotide of the invention can hybridize to the coding sequenceor the complement of the coding sequence of the specified sequence (anyof SEQ ID NOs: 7-12) at a level significantly above background.Background hybridization may occur, for example, because of other DNAspresent in a DNA library. The signal level generated by the interactionbetween a polynucleotide of the invention and the coding sequence orcomplement of the coding sequence of the specific sequence is typicallyat least 10 fold, preferably at least 100 fold, as intense asinteractions between other polynucleotides and the coding sequence ofthe specific sequence. The intensity of interaction may be measured, forexample, by radiolabelling the probe, e.g. with ³²P. Selectivehybridisation may typically be achieved using conditions of medium tohigh stringency. However, such hybridisation may be carried out underany suitable conditions known in the art (see Sambrook et al, 1989) Forexample, if high stringency is required suitable conditions include from0.1 to 0.2×SSC at 60° C. up to 65° C. If lower stringency is requiredsuitable conditions include 2×SSC at 60° C.

The coding sequence of any of SEQ ID NOs: 7-12 may be modified bynucleotide substitutions, for example from 1, 2 or 3 to 10, 25 or 50substitutions. The polynucleotide of any of SEQ ID NOs: 7-12 mayalternatively or additionally be modified by one or more insertionsand/or deletions and/or by an extension at either or both ends.Additional sequences such as signal sequences may also be included.Degenerate substitutions may be made and/or substitutions may be madewhich would result in a conservative amino acid substitution when themodified sequence is translated, for example as shown in the Tableincluded in the Variants section above.

A nucleotide sequence which is capable of selectively hybridizing to thecomplement of the DNA coding sequence of any of SEQ ID NOs: 7-12 willgenerally have at least 70%, at least 80%, at least 90%, at least 95%,at least 98% or at least 99% sequence identity to the specific codingsequence over a region of at least 20, for example at least 30, forinstance at least 40, at least 60, 80, 100 for instance 100 or 200 ormore nucleotides or most preferably over the full length of the specificcoding sequence

For example the UWGCG Package provides the BESTFIT program which can beused to calculate homology (for example used on its default settings)(Devereux et al (1984) Nucleic Acids Research 12, p 387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (typically on their default settings), for example asdescribed in Altschul (1993) J. Mol. Evol. 36:290-300; Altschul et al(1990) J. Mol. Biol. 215:403-10.

Software for performing BLAST analyses is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, 1990). These initial neighbourhoodword hits act as seeds for initiating searches to find HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Extensions for the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, anda comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5877. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to the second sequenceis less than about 1, preferably less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

Any combination of the above mentioned degrees of sequence identity andminimum sizes may be used to define polynucleotides of the invention,with the more stringent combinations (i.e. higher sequence identity overlonger lengths) being preferred. Thus, for example a polynucleotidewhich has at least 90% sequence identity over 25, preferably over 30nucleotides forms one aspect of the invention, as does a polynucleotidewhich has at least 95% sequence identity over 40 nucleotides.

Fragments of polynucleotides may be for example, up to 40, or up to 30nucleotides in length. Preferably the length is up to 5, 10, 15, 20 or25 nucleotides.

In one embodiment, a polynucleotide encoding an agent of the inventionmay comprise any of SEQ ID NOS: 21 to 27. Thus a polynucleotide mayencode NT-proXNP1, NT-proXNP2, NT-proXNP3, NT-proXNP4, NT-proXNP5,proXNP6 or XNP7.

A polynucleotide may comprise:

(a) SEQ ID NO 21, 22, 23, 24, 25, 26 or 27;

(b) a sequence complementary to (a);

(c) a sequence which hybridises under stringent conditions to (a) or(b);

(d) a sequence which is degenerate as a result of the genetic code to(a), (b) or (c);

(e) a sequence having at least 70% identity to any of the sequences in(a) to (d); or

(f) a fragment of any of the sequences in (a) to (e).

Polynucleotides according to the invention may, be producedrecombinantly, synthetically, or by any means available to those ofskill in the art. They may also be cloned by standard techniques. Thepolynucleotides are typically provided in isolated and/or purified form.

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art,

Although in general the techniques mentioned herein are well known inthe art, reference may be made in particular to Sambrook et al,Molecular Cloning: A Laboratory Manual, 1989.

The polynucleotides according to the invention have utility inproduction of the polypeptide agents according to the invention, whichmay take place in vitro, in vivo or ex viva. The polynucleotides may beused in recombinant protein synthesis. Recombinant protein expressionmethods are known in the art and are discussed further below.

Polynucleotides or primers of the invention may carry a revealing label.Suitable labels include radioisotopes such as ¹²⁵I, ³²P or ³⁵S, enzymelabels, or other protein labels such as biotin. Such labels may be addedto polynucleotides or primers of the invention and may be detected usingtechniques known per se.

(c) Vectors, Host Cells and Expression of Peptide Agents

The polynucleotides of the invention may be incorporated into arecombinant replicable vector. The vector may be used to replicate thenucleic acid in a compatible host cell. Therefore, polynucleotides ofthe invention may be made by introducing a polynucleotide of theinvention into a replicable vector, introducing the vector into acompatible host cell and growing the host cell under conditions whichbring about replication of the vector.

In one aspect the vector is an expression vector comprising a nucleicacid sequence that encodes a polypeptide agent of the invention. Suchexpression vectors are routinely constructed in the art of molecularbiology and may, for example, involve the use of plasmid DNA andappropriate initiators, promoters, enhancers and other elements, whichmay be necessary, and which are positioned in the correct orientation,in order to allow for protein expression. Other suitable vectors wouldbe apparent to persons skilled in the art. By way of further example inthis regard we refer to Sambrook et al. 1989.

In one embodiment, a polynucleotide of the invention or for use in theinvention in a vector is operably linked to a control sequence which iscapable of providing for the expression of the coding sequence by thehost cell, i.e. the vector is an expression vector. The term “operablylinked” refers to a juxtaposition wherein the components described arein a relationship permitting them to function in their intended manner.A regulatory sequence, such as a promoter, “operably linked” to a codingsequence is positioned in such a way that expression of the codingsequence is achieved under conditions compatible with the regulatorysequence.

The vectors may be for example, plasmid, virus or phage vectors providedwith a origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.The vectors may contain one or more selectable marker genes, for examplean ampicillin resistance gene in the case of a bacterial plasmid or aresistance gene for a fungal vector.

Promoters and other expression regulation signals may be selected to becompatible with the host cell for which expression is designed. Forexample, yeast promoters include S. cerevisiae GAL4 and ADH promoters,S. pombe nmt1 and adh promoter. Mammalian promoters include themetallothionein promoter which can be induced in response to heavymetals such as cadmium. Viral promoters such as the SV40 large T antigenpromoter or adenovirus promoters may also be used. All these promotersare readily available in the art.

Mammalian promoters, such as β-actin promoters, may be used.Tissue-specific promoters may be used. Viral promoters may also be used,for example the Moloney murine leukaemia virus long terminal repeat(MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSVpromoters (such as the HSV IE promoters), or HPV promoters, particularlythe HPV upstream regulatory region (URR). Viral promoters are readilyavailable in the art.

Vectors may be used in vitro, for example for the production of DNA orRNA or used to transfect or transform a host cell, for example, amammalian host cell.

Expression vectors may be transformed into a suitable host cell toprovide for expression of a polypeptide agent of the invention or apeptide component of agent according to the invention. The host cell,transformed or transfected with an expression vector as described above,is cultivated under conditions to allow for expression of thepolypeptide or fragment, and the expression product is recovered. Thepolypeptide may be isolated and purified using methods known in the art.For example, phage display techniques may be used. Host cells will bechosen to be compatible with the vector and will preferably bebacterial. Host cells may also be cells of a non-human animal, or aplant transformed with a polynucleotide of the invention.

General

Any of the agents, polypeptides, polynucleotides, vectors, cells orantibodies of the invention may be present in substantially isolatedform. They may be mixed with carriers or diluents which will notinterfere with their intended use and still be regarded as substantiallyisolated. They may also be in substantially purified form, in which casethey will generally comprise at least 90%, e.g. at least 95%, 98% or 99%of the proteins, polynucleotides, cells or dry mass of the preparation.

Any of the agents, or antibodies of the invention may be labelled,generally with a suitable detectable label. For example, suitable labelsinclude radiolabels, enzyme labels (e.g. alkaline phosphatase andperoxidase e.g. HRP), chemical labels such as biotin (which may bedetected by avidin or streptavidin conjugated to peroxidase),lanthanides such as europium and fluorescent labels (e.g. fluoresceinand rhodamine), and luminescent or chemiluminescent labels (e.g.acridinium ester, luminol), gold (or other colloid metal) a dye or aparticle. Enzyme labels may be detected using a chemiluminescence orchromogenic based system.

Diagnostics and Monitoring Treatment

The present methods are useful for assessing cardiac health in anindividual. In particular, the methods may be used to detect and assesscardiac impairment. A particular example of cardiac disease that may betargeted using the present methods is congestive heart failure.

Heart failure is a clinical condition characterised by the inability ofthe heart to generate a cardiac output sufficient to meet the demands ofthe body resulting in an activation of the ANP and BNP hormonal systems.Activation of the ANP system is initially associated mainly with atrialoverload whereas activation of the BNP system is primarily suggestive ofventricular overload. Inactivation of the systems is a result of eitherthe patient's own regulatory systems or the use of therapeutic drugs fortreatment of heart failure.

As above, the present methods, by determining the combined levels ofpeptides derived from proANP and proBNP in a sample relative to areference peptide level, can be used to determine activation orinactivation of both the ANP and BNP hormonal systems in an individual.Thus, the present methods may be used to assess functioning of thecardiac systems. The methods of the invention are useful for screeningand ruling out of, assessment of severity of, assessment of prognosis,follow up of treatment and guidance of treatment of cardiac disease suchas heart failure in patients with cardiac pressure or volume overload.

For example, the methods may be used for diagnosis of cardiac disease.The methods may be used to screen individuals, to assess the severity ofcardiac condition, to assess prognosis or to gauge susceptibility to,for example cardiac failure. The present methods may also be employed asa follow-up to treatment for cardiac disease and to assess, monitor andguide treatment of cardiac disease. By monitoring activation orinactivation of the ANP and BNP systems according to the presentmethods, it is possible to assess the effects of treatment in patientssuffering heart disease, for example pharmacological therapy. Thus thepresent methods may be used to assess patient responsiveness to aparticular therapy and to improve the treatment which is provided.

The present methods may be used to assess susceptibility to cardiacdisease. Individuals may then be advised on lifestyle changes which maybe required to decrease the likelihood of developing or decrease theseverity of symptoms associated with cardiac disease such as heartfailure. Individuals may be treated prophylactically for the samepurpose.

Diagnostic Kits

The invention also provides a diagnostic kit. The kit is suitable foruse in the present methods and is in general useful for diagnosis andassessment of cardiac condition as described above.

The contents of the kit will be suitable for the assay format that thekit is intended for. Typically the kit comprises a first bindingsubstance as defined herein, and optionally means for detecting bindingcomplexes formed by the first binding substance, also as describedherein. A kit may additionally comprise an agent according to theinvention, the agent being able to bind to the first binding substancein the kit. The first binding substance and/or the agent may belabelled.

In general a kit may comprise other reagents or components for use inthe particular assay, such as buffers, precipitators, labelling and/ordetection means. In one embodiment the kit will include instructionmeans, such as a package insert instructing the user of the kit as tothe kit contents and assay format.

Thus, a kit for a competitive assay may comprise:

-   -   (a) a first binding substance;    -   (b) labelled agent (NT-proXNP, proXNP or XNP);    -   (c) a standard (NT-proXNP, proXNP or XNP); and    -   (d) other usual materials according to the detection system, eg.        precipitators, buffers etc.        A kit for a sandwich assay may comprise:    -   (a) first binding substance;    -   (b) labelled second binding substance;    -   (c) a standard (NT-proXNP, proXNP, XNP);    -   (c) other usual materials according to the detection system.

EXAMPLES

The following Examples illustrate the invention. Unless indicatedotherwise, the methods used are standard biochemistry and molecularbiology techniques. Examples of suitable general methodology textbooksinclude Sambrook et al, Molecular Cloning, A Laboratory Manual (1989)and Ausubel et al, Current Protocols in Molecular Biology (1995) JohnWiley and Sons Inc.

Example 1 Expression and Purification of Recombinant NT-proXNP

The nucleotides encoding amino acids 1-37 of human NT-proBNP and thoseencoding amino acids 29-98 of human NT-proANP are amplified by reversetranscription PCR from human atrial RNA using oligonucleotide primers.The 5′-primer for NT-proBNP amplification contains the cleavage site forthe restriction enzyme BamHI (5′-GCGGATCCCACCCGCTGGGCAGCCCCG-3′ Seq IDNO:28) and the 3′-primer for XbaI (5′-GCTCTAGAGGATGTCTGCTCCACC-3′ SEQ IDNO:29). The 5′-primer for NT-proANP amplification has XbaI linker(5′-GCTCTAGAGAAGATGAGGTCGTGC-3′ SEQ ID NO:30) and the 3′-primer hasEcoRI linker (5′-GCGAATTCTCACCGAGGGGCAGTGAGC-3′ SEQ ID NO:31). Inaddition, the NT-proANP amplicon contains an in-frame termination codon(TGA) at its 3′-end preceding the EcoRI cleavage site. The other versionof NT-proBNP amplicon contains an in-frame codon for Tyr at its5′-terminus following the BamHI linker sequence(5′-GCGGATCCTACCACCCGCTGGGCAG-3′ SEQ ID NO:32). The RT-PCR products, arepurified by agarose electrophoresis, cleaved with XbaI and BamHI orEcoRI and subcloned end-to-end (NT-proBNP->NT-proANP) into BamHI/EcoRIsite of pGEX-4T-1 vector (Amersham Pharmacia Biotech, Uppsala, Sweden).The nucleotide sequences and reading frames of the constructs areconfirmed by sequencing.

The expression and affinity purification of the GST-proteins are carriedout according to the following procedure. An overnight culture of E.coli, transformed with a recombinant plasmid, is diluted 1:100 in 2×YTAand grown at 37° C. until the OD at 660 nm reaches 0.6.Isopropyl-1-thio-D-galactopyranoside (IPTG) is added to a finalconcentration of 0.1 mM and the culture is further incubated for 1-2 h.The bacterial cells are harvested by centrifugation (7000 g for 10 min.at +4° C.), resuspended in PBS (50 μl/ml of culture) and sonicated. Thecell lysate is cleared at 7000 g for 15 min. The supernatant is appliedto a glutathione agarose column (Sigma, Saint Louis, Mo., USA) andwashed three times with PBS. The fusion protein is eluted with 10 mMglutathione in 50 nM Tris-HCl, pH 8.0 and stored in aliquots at 20° C.Samples are separated by SDS-PAGE (12% acrylamide). Both prokaryotic oreukaryotic expression vectors can be used. Accordingly, the wholepeptide or at least a portion of said peptide or protein may be producedin prokaryotic or eukaryotic cells.

The recombinant peptides are released from fusion partner by treatingwith thrombin (Amersham Pharmacia Biotech) at room temperature for 1 h(1 U/100 μg protein). The peptides are purified by reverse-phase HPLCusing a 4.6×150 mm Vydac C₄ column. The column is eluted with a linear40 min gradient from 20-48% acetonitrile in aqueous trifluoroaceticacid. Elution rate is 1 ml/min and absorbance at 200-280 nm is measuredduring HPLC to monitor the purity of products.

An example of the HPLC profile of the purified product consisting of(from NH₂ to CO₂H-terminus) human proBNP₁₋₃₇, a short spacer, serine andarginine, and human proANP₂₉₋₉₈, is presented in FIG. 1. Two additionalamino acids, glycine and serine, originated from GST are left in theN-terminus of the peptide as an adduct.

Example 2 Chemical Synthesis of NT-proXNP

The combination epitope NT-proXNP5 comprising (from NH₂— to CO₂Hterminus) the sequences human proBNP₁₀₋₂₉, Cys spacer and humanproANP₆₀₋₈₀ was assembled with a Peptide Synthesizer using Fmocchemistry. Alternatively the combination epitope peptide NT-proXNP1comprising (from NH₂- to CO₂H-terminus) the sequences human proBNP₁₅₋₂₄,Gly-Lys-Tyr-Gly spacer and human proANP₈₂₋₉₆, was assembled. The productwas cleaved from the HMP resin with 95% trifluoroacetic acid/2.5% H₂O,2.5% tri-isopropylsilane, precipitated with diethyl ether, dried anddesalted on Sephadex G-15 in 30% acetic acid. The peptide was purifiedby reverse phase HPLC in a preparative RCM NovaPak C₁₈ cartridge (2.5×10cm) with a linear gradient of acetonitrile in aqueous 0.1%trifluoroacetic acid. The purity was ascertained by reverse phase HPLCin elution conditions with different selectivity. The identity of thepeptide was confirmed by amino acid analysis or MALDI-TOF massspectrometry and peptide mapping.

Example 3 Immunoassay of NT-proXNP

Binding substance was prepared from goat antibodies obtained by using asimmunogen affinity purified, fusion protein of GST/NT-proANP₂₀₋₈₀ andNT-proBNP₁₀₋₂₉-TBG or GST-fusion protein of NT-pro-BNP₁₋₃₇ andNT-proANP₂₉₋₉₈. The latter of the peptide antigens was prepared with themethods described in Example 1 and contains human proBNP₁₋₃₇, Ser-Argspacer, human proANP₂₉₋₉₈ and Gly-Ser adduct. Alternatively, anotherpeptide antigen was prepared with the methods described in Example 2comprising (from NH₂— to CO₂H terminus) the sequences human proBNP₁₀₋₂₉,cysteine spacer from which it was coupled to bovine thyroglobulin (TBG)prior to immunisation and human proANP₆₀₋₈₀. Goats were injected atmultiple sites at the back with 1-1.5 mg of immunogen in 1 ml 0.9% NaClemulsified in equal volume of Freund's complete adjuvant. Boosters of0.5 mg in Freund's incomplete adjuvant were given 2-4 times at 2-3 weeksintervals and blood is drawn 14 days after the fast injections. Theantisera were chosen according to the titre of binding theradioiodinated peptide or protein agent of the invention (see below), aswell as the sensitivity and specificity with regard to related peptidesand peptide or protein agent of the invention. Any modification of thepeptide or protein agent of the invention or any fragment or derivativethereof may also be used for immunisation purposes to produce eithermonoclonal or polyclonal antibodies.

Recombinant NT-proBNP₁₋₃₇/NT-proANP₂₉₋₉₈ (1.5 μg), produced as describedin Example 1, was radioiodinated using 0.5 mCi Na¹²⁵I in the presence of10 μg chloramine-T in 0.5 M phosphate buffer, pH 7.5 for 60 sec.,followed by the addition of 10 μg sodium disulphite. The mixture wasdesalted by Sephadex G-25 gel filtration and purified by reverse phaseHPLC in a Symmetry C₁₈ column and a 30 min linear 20% to 50%acetonitrile gradient in aqueous trifluoroacetic acid at a flow rate of1 ml/min. Fractions of 1 ml were collected and monitored forradioactivity in a Multi-Gamma counter (Wallac, Turku, Finland).

Recombinant NT-proBNP₁₋₃₇/NT-proANP₂₉₋₉₈, produced as described inExample 1, was used also as the assay calibrator in the NT-proXNPimmunoassay. The assay buffer used for all dilutions consists of 0.04 Msodium hydrogen phosphate, 0.01 M sodium dihydrogen phosphate, 0.1 MNaCl, 0.1% gelatine, 0.05% Triton X-100, pH 7.4). Plasma or serumsamples were incubated in duplicates of 25 μl with 100 μl of antiserumand 100 μl of tracer solution (containing approx. 8 000 cpm of iodinatedpeptide) for 16-24 h at +4° C. Calibration was performed by incubatingcalibrators (0.08-8 nmol l⁻¹) with the same amount and concentration ofantiserum, tracer and anti-antiserum for the same time period as above.The amount of antiserum assayed was determined to bind 40-50% of thetracer when no competitor was present, in order to ensure sufficientcompetition in binding.

FIG. 2 b shows development of antibody titres in immunisation of a goatusing GST-fusion protein of NT-proBNP₁₋₃₇/NT-proANP₂₉₋₉₈ (comprising SEQID NO: 14) as immunogen, titres after 1^(st), 3^(rd) and 4^(th) boosterin RIA of NT-proANP₁₋₉₈ and NT-proBNP₁₋₇₆. FIG. 2 b shows that, forexample, a binding substance prepared in a goat, obtained by usingGST-fusion protein of NT-proBNP₁₋₃₇/NT-proANP₂₉₋₉₈ (comprising SEQ IDNO: 14) as immunogen, typically in dilution of 1:50,000-1:60,000 wassuitable for 40-50% binding and simultaneous binding of NT-proANP (SEQID NO: 3) and NT-proBNP (SEQ ID NO: 6) demonstrated also in separateradioimmunoassay of NT-proANP₁₋₉₈ and NT-proBNP₁₋₇₆. A similar bindingsubstance was produced in immunisation of a goat using TBG-conjugate ofNT-proBNP₁₀₋₂₉/NT-proANP₆₀₋₈₀, (comprising SEQ ID NO: 18) as immunogen.A typical dilution in competitive NT-proXNP assay was ranging from1:10,000 to 1:15,000.

Bound and free NT-proXNP were separated by precipitation with donkeyanti-goat IgG in 0.5 ml of 8% polyethylene glycol 6000, containingnormal goat serum carrier (1 μl). After centrifugation, the pellet wascounted for radioactivity. An example of a reference curve obtained bythis type of assay is presented in FIG. 2 a.

FIG. 2 a shows a competitive binding curve for immunoassay of NT-proXNP.The assay utilises recombinant NT-proBNP₁₋₃₇/NT-proANP₂₉₋₉₈ ascalibrator and tracer and polyclonal goat antibody based bindingsubstance to recognise NT-proXNP, NT-proANP and NT-proBNPsimultaneously. The X-axis depicts the amount of calibrator added andthe Y-axis Bound/Bound with no calibrator added.

The immunoassay of NT-proXNP described in Example 3 was used todetermine the serum levels of NT-proXNP in 700 patients with cardiacdisorders. The results are shown in FIG. 3. The levels of NT-proXNP arehighly significantly correlating with NT-proANP and NT-proBNP levelsmeasured from the same samples by separate in-house radioimmunoassays ofNT-proANP₁₋₉₈ and NT-proBNP₁₋₇₆.

The methods of Example 3 were used to assay the serum levels ofNT-proXNP, in 500 cardiac patients classified according to the New YorkHeart Association (NYHA) scale. The results are shown in FIG. 4. Theserum levels of NT-proANP and NT-proBNP measured by separate in-houseradioimmunoassays from the same samples are displayed for reference asmeasure of activation ANP and BNP systems.

The methods of Example 3 were used to assay plasma levels of NT-proXNPin patients suffering heart failure and these were correlated withpositive effect of pharmacological therapy in the patients. Results areshown in FIG. 5. The serum levels of NT-proANP and NT-proBNP measuredfrom the same samples by separate in-house radioimmunoassays aredisplayed for reference. Patients (n=11) suffering from heart failure(stabile NYHA class II-III) were treated by intravenous infusion of aninodilatator for 24 hours. Cardiac output (CO) as ml/min was measuredwith echocardiography. The levels of NT-proANP, NT-proBNP and NT-proXNPwere assayed before and at time point of 24 hours from the start ofadministration of the drug. The relative sensitivity to detect responseto the treatment was determined at the cut-off levels of 10% increase inCO, as determined with echocardiography and 20% decrease in NT-ProANPand NT-ProBNP and NT-proXNP as measure of inactivation of ANP and BNPsystems. NT-proXNP exceeded cut-off in all of 11 cases, whereasNT-proANP and NT-proBNP and CO excluded cut off in 9 of 11 cases.

REFERENCES

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1. An in vitro method of determining activation or inactivation of theatrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP)hormonal systems in a subject, the method comprising detecting in asingle reading, in a single assay the combined presence of atrial andbrain natriuretic peptide prohormones (proANP and proBNP) or fragmentsthereof in a sample from the subject, wherein, compared to a referencelevel, detection of an increase in the combined presence of proANP andproBNP, or fragments thereof, in the sample indicates activation of theANP and BNP hormonal systems, and wherein, compared to a referencelevel, detection of a decrease in the combined presence of proANP andproBNP, or fragments thereof, in the sample indicates inactivation ofthese systems, wherein said method does not comprise detection of thepresence of proANP and proBNP or fragments thereof individually; whereindetection of activation of the ANP and BNP hormonal systems isdiagnostic of cardiac impairment or detection of inactivation of ANP andBNP hormonal systems indicates successful treatment of cardiacimpairment.
 2. The method according to claim 1, which comprisescontacting the sample with a bi- or oligo-specific first bindingsubstance that is able to bind to: (a) proANP (SEQ ID NO:1), ANP (SEQ IDNO:2), or NT-proANP (SEQ ID NO:3); and (b) proBNP (SEQ ID NO:4), BNP(SEQ ID NO:5), or NT-proBNP (SEQ ID NO:6).
 3. The method according toclaim 1 which comprises contacting the sample with a fusion polypeptideagent comprising both: (a) proANP (SEQ ID NO:1), ANP (SEQ ID NO:2), orNT-proANP (SEQ ID NO:3); and (b) proBNP (SEQ ID NO:4), BNP (SEQ IDNO:5), or NT-proBNP (SEQ ID NO:6); wherein said fusion polypeptide agentcan be bound by a first binding substance, and said fusion polypeptideagent is used as a calibration agent or a competitive inhibitor; andsaid first binding substance, which is able to bind to: (c) proANP (SEQID NO:1), ANP (SEQ ID NO:2), or NT-proANP (SEQ ID NO:3); (d) proBNP (SEQID NO:4), BNP (SEQ ID NO:5), or NT-proBNP (SEQ ID NO:6); and (e) saidfusion polypeptide agent.
 4. The method according to claim 3 wherein thefirst binding substance comprises: (a) a bi- or oligo-specific bindingsubstance; or (b) a mixture of mono-specific binding substances.
 5. Themethod according to claim 2 wherein the first binding substancecomprises an antibody or a fragment or derivative thereof, wherein saidfragment or derivative thereof retains the binding characteristics ofsaid antibody.
 6. The method according to claim 5 wherein the antibodycomprises a polyclonal antibody, monoclonal antibody, oligoclonalantibody, bifunctional antibody or crossreacting polyclonal antibody. 7.The method according to claim 3 wherein in the fusion polypeptide agent,(a) is SEQ ID NO:3 and (b) is SEQ ID NO: 6, or (a) is SEQ ID NO:2 and(b) is SEQ ID NO:5.
 8. The method according to claim 1 which comprisescontacting the sample with a fusion polypeptide agent comprising: (a)proBNP₁₅₋₂₄ and proANP₈₂₋₉₆; (b) proBNP₁₋₃₇ and proANP₂₉₋₉₈; (c)proBNP₁₀₋₂₉ and proANP₂₀₋₈₀; (d) proBNP₁₋₇₆ and proANP₁₋₉₈; (e)proBNP₁₀₋₂₉ and proANP₆₀₋₈₀; (f) proBNP₁₋₁₀₈ and proANP₁₋₁₂₆; or (g)proBNP₇₇₋₉₂ and proANP₁₁₂₋₁₂₆; wherein said fusion polypeptide agent canbe bound by a first binding substance, and said fusion polypeptide agentis used as a calibration agent or a competitive inhibitor; and saidfirst binding substance, which is able to bind to: (h) proANP (SEQ IDNO:1), ANP (SEQ ID NO:2), or NT-proANP (SEQ ID NO:3); and (i) proBNP(SEQ ID NO:4), BNP (SEQ ID NO:5), or NT-proBNP (SEQ ID NO:6).
 9. Themethod according to claim 2 wherein the first binding substance and/orthe fusion polypeptide agent is: (a) labelled with a detectable label;and/or (b) immobilised.
 10. The method according to claim 2 whichadditionally comprises contacting the sample with a second bindingsubstance which is able to bind to the first binding substance.
 11. Themethod according to claim 10 wherein the second binding substance is:(a) labelled with a detectable label; and/or (b) immobilised.
 12. Themethod according to claim 10 wherein the second binding substance causesprecipitation of the first binding substance and any peptide which isbound to it.
 13. The method according to claim 1 which comprises animmunoassay.
 14. The method of claim 1, wherein said reference level isdetermined from a previous measurement from said subject.
 15. The methodof claim 1, wherein said reference level is based on the normal level ofa population of subjects.
 16. The method of claim 15, wherein saidpopulation of subjects is the general population.
 17. The method ofclaim 1, wherein said assay is calibrated so that a particular readingin the assay is known to represent the normal peptide level.
 18. Themethod of claim 1, wherein said assay is calibrated so that a normallevel will produce a negligible or insignificant result.
 19. The methodof claim 3, wherein said assay is calibrated by use of said fusionpolypeptide agent.
 20. The method of claim 1, wherein said subject is ahuman.